Production of M-/GM-group aflatoxins catalyzed by the OrdA enzyme in aflatoxin biosynthesis.

Aspergillus parasiticus produces the minor aflatoxins M(1) (AFM(1)), M(2) (AFM(2)), GM(1) (AFGM(1)), and GM(2) (AFGM(2)), as well as the major aflatoxins B(1) (AFB(1)), B(2) (AFB(2)), G(1) (AFG(1)), and G(2) (AFG(2)). Feeding of A. parasiticus with aspertoxin (12c-hydroxyOMST) caused AFM(1) and AFGM(1), and cell-free experiments using the microsomal fraction of A. parasiticus and aspertoxin caused production of AFM(1), indicating that aspertoxin is a precursor of AFM(1) and AFGM(1). Feeding of the same fungus with O-methylsterigmatocystin (OMST) caused AFM(1) and AFGM(1) together with AFB(1) and AFG(1); feeding with dihydroOMST (DHOMST) caused AFM(2) and AFGM(2) together with AFB(2) and AFG(2). Incubation of either the microsomal fraction or OrdA enzyme-expressing yeast with OMST caused production of aspertoxin together with AFM(1) and AFB(1). These results demonstrated that the OrdA enzyme catalyzes both 12c-hydroxylation reaction from OMST to aspertoxin and the successive reaction from aspertoxin to AFM(1). In contrast, feeding of the fungus with AFB(1) did not produce any AFM(1), demonstrating that M-/GM-aflatoxins are not produced from B-/G-aflatoxins. Furthermore, AFM(1) together with AFB(1) and AFG(1) was also produced from 11-hydroxyOMST (HOMST) in feeding experiment of A. parasiticus, whereas no aflatoxins were produced when used the ordA deletion mutant. These results demonstrated that OrdA enzyme can also catalyze 12c-hydroxylation of HOMST to produce 11-hydroxyaspertoxin, which serves as a precursor for the production of AFM(1) and AFGM(1). The same pathway may work for the production of AFM(2) and AFGM(2) from DHOMST and dihydroHOMST through the formation of dihydroaspertoxin and dihydro-11-hydroxyaspertoxin, respectively.

Clinical features of hemichoreahemiballism: A stroke-related movement disorder.

We examined pathogenesis and clinical features of three hemichorea-hemiballism (HCHB) cases. We studied their age, magnetic resonance imaging results, vascular risk factors, management, and outcomes. One man and two women (aged 74-86 years) demonstrated acute onset of HCHB, lasting for at least several months. Patients had one or more vascular risk factors, including hypertension and diabetes. All patients presented subacute or old infarction in the basal ganglia with contralateral symptoms. We administered clonazepam (0.5-1 mg/day), haloperidol (0.375-0.75 mg/day), or both as necessary and observed symptom-control. Vascular lesions in the basal ganglia were a contributing factor. Symptoms were controlled using pharmacotherapy with gamma-aminobutyric acid-agonist (clonazepam) or anti-dopaminergic (haloperidol) medication.

Beta-synemin expression in cardiotoxin-injected rat skeletal muscle.

BACKGROUND: Beta-synemin was originally identified in humans as an alpha-dystrobrevin-binding protein through a yeast two-hybrid screen using an amino acid sequence derived from exons 1 through 16 of alpha-dystrobrevin, a region common to both alpha-dystrobrevin-1 and -2. alpha-Dystrobrevin-1 and -2 are both expressed in muscle and co-localization experiments have determined which isoform preferentially functions with beta-synemin in vivo. The aim of our study is to show whether each alpha-dystrobrevin isoform has the same affinity for beta-synemin or whether one of the isoforms preferentially functions with beta-synemin in muscle. METHODS: The two alpha-dystrobrevin isoforms (-1 and -2) and beta-synemin were localized in regenerating rat tibialis anterior muscle using immunoprecipitation, immunohistochemical and immunoblot analyses. Immunoprecipitation and co-localization studies for alpha-dystrobrevin and beta-synemin were performed in regenerating muscle following cardiotoxin injection. Protein expression was then compared to that of developing rat muscle using immunoblot analysis. RESULTS: With an anti-alpha-dystrobrevin antibody, beta-synemin co-immunoprecipitated with alpha-dystrobrevin whereas with an anti-beta-synemin antibody, alpha-dystrobrevin-1 (rather than the -2 isoform) preferentially co-immunoprecipitated with beta-synemin. Immunohistochemical experiments show that beta-synemin and alpha-dystrobrevin co-localize in rat skeletal muscle. In regenerating muscle, beta-synemin is first expressed at the sarcolemma and in the cytoplasm at day 5 following cardiotoxin injection. Similarly, beta-synemin and alpha-dystrobrevin-1 are detected by immunoblot analysis as weak bands by day 7. In contrast, immunoblot analysis shows that alpha-dystrobrevin-2 is expressed as early as 1 day post-injection in regenerating muscle. These results are similar to that of developing muscle. For example, in embryonic rats, immunoblot analysis shows that beta-synemin and alpha-dystrobevin-1 are weakly expressed in developing lower limb muscle at 5 days post-birth, while alpha-dystrobrevin-2 is detectable before birth in 20-day post-fertilization embryos. CONCLUSION: Our results clearly show that beta-synemin expression correlates with that of alpha-dystrobrevin-1, suggesting that beta-synemin preferentially functions with alpha-dystrobrevin-1 in vivo and that these proteins are likely to function coordinately to play a vital role in developing and regenerating muscle.

Clathrin isoform CHC22, a component of neuromuscular and myotendinous junctions, binds sorting nexin 5 and has increased expression during myogenesis and muscle regeneration.

The muscle isoform of clathrin heavy chain, CHC22, has 85% sequence identity to the ubiquitously expressed CHC17, yet its expression pattern and function appear to be distinct from those of well-characterized clathrin-coated vesicles. In mature muscle CHC22 is preferentially concentrated at neuromuscular and myotendinous junctions, suggesting a role at sarcolemmal contacts with extracellular matrix. During myoblast differentiation, CHC22 expression is increased, initially localized with desmin and nestin and then preferentially segregated to the poles of fused myoblasts. CHC22 expression is also increased in regenerating muscle fibers with the same time course as embryonic myosin, indicating a role in muscle repair. CHC22 binds to sorting nexin 5 through a coiled-coil domain present in both partners, which is absent in CHC17 and coincides with the region on CHC17 that binds the regulatory light-chain subunit. These differential binding data suggest a mechanism for the distinct functions of CHC22 relative to CHC17 in membrane traffic during muscle development, repair, and at neuromuscular and myotendinous junctions.

A case of complex partial seizure with reversible MRI abnormalities in the elderly.

A 79-year-old woman was admitted to our hospital because of prolonged impaired consciousness and right hemiparesis. She was treated for acute cerebral infarction because her brain magnetic resonance imaging showed extensive cortical lesions similar to acute infarction in diffusion weighted image, fluid attenuated inversion recovery, and T2 weighted images. On the fifth day, she had a focal seizure on the right side. A new lesion during imaging and electroencephalogram abnormality were observed at that time. After the antiepileptic drug treatment was started, her right hemiparesis considered as ictal paresis, confusion, and the magnetic resonance imaging findings gradually improved. There was also an old, irreversible lesion in the left hippocampus, which was considered as the focus of her complex partial seizure. In the elderly, the post-ictal period of confusion, which occurs with complex partial seizure, may be prolonged. In our case, improvement of hemiparesis and confusion occurred after about 2 weeks.

The expression of dystrophin, alpha-sarcoglycan, and beta-dystroglycan during skeletal muscle regeneration: immunohistochemical and western blot studies.

We evaluated re-expression of dystrophin, alpha-sarcoglycan and beta-dystroglycan in regenerating skeletal muscles of rats after cardiotoxin-induced myonecrosis in order to understand the dynamic behaviour of these proteins during the regeneration process. Immunohistochemical staining of these proteins almost disappeared in the sarcolemma of necrotic fibers on the 1st day, and was obscured due to non-specific staining on the 3rd day. Dystrophin was labeled faintly at the sarcolemma of regenerating muscle fibers on the 5th day. From the 5th day to the 10th day, levels of immunostaining of dystrophin increased. After the 14th day, dystrophin was stained conspicuously. alpha-Sarcoglycan was labeled weakly at the sarcolemma of small regenerating muscle fibers on the 5th day and was labeled conspicuously after the 7th day. beta-Dystroglycan was labeled moderately at the sarcolemma of regenerating muscle fibers on the 5th day and was labeled conspicuously after the 7th day. In western blot analysis, beta-dystroglycan persisted throughout the entire cycle of myonecrosis and regeneration, and re-expression of alpha-sarcoglycan progressed faster than that of dystrophin. We speculate that regeneration advances from the basement membrane side to the subsarcolemmal side, and that proteins at the basement membrane side resist disruption and have a high capacity for regeneration.

The CHC22 clathrin-GLUT4 transport pathway contributes to skeletal muscle regeneration.

Mobilization of the GLUT4 glucose transporter from intracellular storage vesicles provides a mechanism for insulin-responsive glucose import into skeletal muscle. In humans, clathrin isoform CHC22 participates in formation of the GLUT4 storage compartment in skeletal muscle and fat. CHC22 function is limited to retrograde endosomal sorting and is restricted in its tissue expression and species distribution compared to the conserved CHC17 isoform that mediates endocytosis and several other membrane traffic pathways. Previously, we noted that CHC22 was expressed at elevated levels in regenerating rat muscle. Here we investigate whether the GLUT4 pathway in which CHC22 participates could play a role in muscle regeneration in humans and we test this possibility using CHC22-transgenic mice, which do not normally express CHC22. We observed that GLUT4 expression is elevated in parallel with that of CHC22 in regenerating skeletal muscle fibers from patients with inflammatory and other myopathies. Regenerating human myofibers displayed concurrent increases in expression of VAMP2, another regulator of GLUT4 transport. Regenerating fibers from wild-type mouse skeletal muscle injected with cardiotoxin also showed increased levels of GLUT4 and VAMP2. We previously demonstrated that transgenic mice expressing CHC22 in their muscle over-sequester GLUT4 and VAMP2 and have defective GLUT4 trafficking leading to diabetic symptoms. In this study, we find that muscle regeneration rates in CHC22 mice were delayed compared to wild-type mice, and myoblasts isolated from these mice did not proliferate in response to glucose. Additionally, CHC22-expressing mouse muscle displayed a fiber type switch from oxidative to glycolytic, similar to that observed in type 2 diabetic patients. These observations implicate the pathway for GLUT4 transport in regeneration of both human and mouse skeletal muscle, and demonstrate a role for this pathway in maintenance of muscle fiber type. Extrapolating these findings, CHC22 and GLUT4 can be considered markers of muscle regeneration in humans.

A role for the CHC22 clathrin heavy-chain isoform in human glucose metabolism.

Intracellular trafficking of the glucose transporter GLUT4 from storage compartments to the plasma membrane is triggered in muscle and fat during the body's response to insulin. Clathrin is involved in intracellular trafficking, and in humans, the clathrin heavy-chain isoform CHC22 is highly expressed in skeletal muscle. We found a role for CHC22 in the formation of insulin-responsive GLUT4 compartments in human muscle and adipocytes. CHC22 also associated with expanded GLUT4 compartments in muscle from type 2 diabetic patients. Tissue-specific introduction of CHC22 in mice, which have only a pseudogene for this protein, caused aberrant localization of GLUT4 transport pathway components in their muscle, as well as features of diabetes. Thus, CHC22-dependent membrane trafficking constitutes a species-restricted pathway in human muscle and fat with potential implications for type 2 diabetes.

The expression of alpha-dystrobrevin and dystrophin during skeletal muscle regeneration.

The expression of alpha-dystrobrevin and dystrophin in rat tibialis anterior muscles was chronologically evaluated during a cycle of regeneration after myonecrosis induced by the injection of cardiotoxin. In immunohistochemical studies, alpha-dystrobrevin and dystrophin were first stained weakly at the sarcolemma of some regenerating muscle fibers on day 5. On day 7, alpha-dystrobrevin was still stained weakly, whereas dystrophin was stained conspicuously. After day 10, alpha-dystrobrevin and dystrophin were both stained conspicuously on almost all regenerating muscle fibers. In the Western blot analysis, alpha-dystrobrevin and dystrophin were first detected as visible bands on days 5 and 7, respectively. The bands of alpha-dystrobrevin and dystrophin both darkened sequentially up to day 10. The protein levels based on the densitometrical analysis of the bands on each day were converted to the percentage of the protein level on day 28, which was taken as 100%. The sequential line based on these data showed that alpha-dystrobrevin and dystrophin reached 50% of the protein level on day 28 by 6.6 and 5.3 days, respectively. These data provide evidence that alpha-dystrobrevin regenerates more slowly than dystrophin in skeletal muscle.

The expression of neuronal nitric oxide synthase and dystrophin in rat regenerating muscles.

We investigated the expression of neuronal nitric oxide synthase (nNOS) and dystrophin in the regenerating skeletal muscles of rats after cardiotoxin-induced myonecrosis by immunohistochemical studies and western blot analysis. In normal muscles, nNOS was moderately immunostained on type 2B fibers, but was faintly immunostained on type 2A or type 1 fibers. In immunohistochemical studies of regenerating muscles, nNOS was first observed at the sarcolemma of type 2B fibers on day 10, when the type discrimination between types 2A and 2B was first detected by ATP reactions. Subsequently, the immunostaining of nNOS grew progressively stronger in type 2B fibers, with faint staining in type 2A and type I fibers until day 28. Meanwhile, the immunostaining of dystrophin grew stronger equally in all three fibers until day 21. In western blot analysis of regenerating muscles, nNOS regenerated more slowly than dystrophin. The present data suggest that the expression of nNOS is related to the muscle fiber type differentiation, and that the role of nNOS is related to the function of the type 2B fibers of the muscle.